BackgroundMitochondrial Ca2+ (mtCa2+) uptake via a main mtCa2+ channel, mtCa2+ uniporter (MCU), is an essential element to produce ATP at the mitochondria, the form of energy indispensable for the development and survival of various cell types including in cardiomyocytes (CMs). However, the excessive amount of mtCa2+ oppositely decreases ATP production by the mitochondria and also initiates apoptotic signaling activation from mitochondria. Our group previously showed that tyrosine phosphorylation of MCU can be responsible for the mtCa2+ overload in the CMs. Specifically, Gq protein–coupled receptor (GqPCR) stimulation promotes activation of a tyrosine kinase named proline‐rich tyrosine kinase 2 (Pyk2), and its translocation from cytosol to the mitochondrial matrix, which leads to the tyrosine phosphorylation of MCU, mtCa2+ overload, and activation of apoptotic signaling in the CMs. Currently, clinical Pyk2 inhibitor is under the clinical trial in Europe for treating several types of cancer, but the strategies/tools to inhibit Pyk2 only in the mitochondria are still not available.HypothesisTargeting Pyk2‐specific inhibitory peptide in the mitochondria can efficiently blocks Pyk2 activation in the mitochondria and mtCa2+ overload GqPCR stimulationMethodsWe added mitochondrial matrix‐targeted sequence to the N‐terminal of Pyk2‐specific inhibitory peptide (C‐terminal portion of Pyk2, also known as Cell Adhesion Kinase‐β‐Related Non‐Kinase, CRNK) to generate mitochondrial targeted CRNK (mt‐CRNK). CRNK and mt‐CRNK were transiently overexpressed in HEK293T cells using mammalian expression vectors. Whole cell lysates were collected and used for biochemical assay including Western blotting.ResultsWe confirmed that transient transfections of the plasmids containing CRNK and mt‐CRNK were successfully yield overexpression of these proteins in HEK293Tcells confirmed by specific antibody against C‐terminal of Pyk2. We also confirmed using confocal microscopy that GFP‐tagged mt‐CRNK is exclusively expressed in the mitochondria. Stimulation of Gq‐coupled alpha1‐adrenergic receptor by phenylephrine was able to activate Pky2, and overexpression of either CRNK or mt‐CRNK significantly inhibited phenylephrine‐mediated Pyk2 activationConclusionsMitochondrial matrix‐targeted Pyk2 inhibitory peptide mt‐CRNK can significantly inhibit Pyk2 activation under GqPCR stimulation. Therefore, the use of mt‐CRNK may prevent mtCa2+ overload and cell death signaling. The outcome of our research will offer us a new therapeutic approaches for various human diseases such as heart failure and heart attack, where mtCa2+ overload plays an important roles for disease formation and progress.Support or Funding InformationApart of this research was supported by the American Physiological Society (APS) Hearst Undergraduate Summer Research Fellowship (to D.A.T.), American Heart Association (AHA) 18CDA34110091(toB.S.J), NIH/NHLBI R01HL136757 (to J.O.‐U.), AHA 16SDG27260248 (to J.O.‐U.), and APS 2017 Shih‐Chun Wang Young Investigator Award.